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PubMed1991

Characterization of the effects of the new inotropic agent BDF 9148 in isolated papillary muscles and myocytes of the guinea-pig heart.

1. The cardiotonic agent BDF 9148 (4-[3'-(1''-benzhydryl-azetidine-3''-oxy)-2'-hydroxypropoxy]-1H-indole- 2-carbonitrile) is structurally related to DPI 201-106 (4-[3'-(4''-benzhydryl-1''-piperazinyl) -2'-hydroxypropoxy]-1H-indole-2-carbonitrile) which is known to modify cardiac sodium channels. In guinea-pig papillary muscles, both compounds increase force of contraction with similar concentration-response curves. Like DPI 201-106, BDF 9148 prolongs the action potential duration in a tetrodotoxin-sensitive manner. With high concentrations (greater than 3 microM), however, the action potential duration shortens again. In order to elucidate the underlying changes in membrane currents, we have investigated the effects of BDF 9148 in isolated ventricular myocytes of the guinea-pig heart. 2. In isolated cells, a concentration of 1 microM BDF 9148 prolonged the action potential duration and markedly enhanced unloaded cell shortening, indicating that the procedure of cell isolation does not abolish the effect of the drug. 3. Membrane currents were studied with the single electrode voltage clamp technique. With clamp steps from -80 mV to -40 mV, BDF 9148 (1 microM) induced a slowly decaying inward current which was suppressed by tetrodotoxin. Therefore, like DPI 201-106, BDF 9148 slows the inactivation of the sodium channels. 4. In order to quantify the effects of BDF 9148 and DPI 201-106 on sodium current inactivation, we have measured the inward current amplitude still present at 100 ms after a depolarizing clamp step from -80 mV to -30 mV. Both drugs increased this current component in a concentration-dependent manner; however, BDF 9148 had a larger effect in the low concentration range.5. The calcium current was inhibited by BDF 9148 and DPI 201-106 in a concentration-dependent manner; the pD2 values were 5.70 and 5.95, respectively.6. The two compounds are thought to produce similar positive inotropic effects by imposing a sodium load on the muscle cells via modification of the sodium channels. The differences in action potential duration could be due to different contributions of ionic currents other than sodium or calcium currents and of pump and exchange currents. At present, there is not sufficient data to identify clearly distinct current components responsible for the differences in action potential prolongation.

Action Potentials↗
PubMed1995

E-4695, a new C-7 azetidinyl fluoronaphthyridine with enhanced activity against gram-positive and anaerobic pathogens.

E-4695, (-)-7-[3-(R)-amino-2-(S)-methyl-1-azetidinyl]-1-cyclopropyl-1,4- dihydro-6-fluoro-4-oxo-1,8-naphthyridine-3-carboxylic acid, is a new fluorinated naphthyridine with an azetidine moiety. The MICs of E-4695 at which 90% of the isolates were inhibited (MIC90s) were 0.06 to 0.5 microgram/ml for gram-positive cocci, including species of the genera Staphylococcus, Streptococcus, and Enterococcus, and the MIC90s against gram-negative pathogens such as members of the family Enterobacteriaceae (with the exception of Providencia spp. [MIC90, 8 micrograms/ml]) and Pseudomonas aeruginosa were 0.015 to 0.5 microgram/ml. E-4695 inhibited 90% of the Clostridium perfringens and Bacteroides fragilis isolates at 0.25 and 4 micrograms/ml, respectively. Against gram-positive cocci the potency of E-4695 was 2- to 8-fold higher than that of ciprofloxacin, 4- to 8-fold higher than that of ofloxacin, and 8- to 16-fold higher than that of fleroxacin. Against enteric bacteria and P. aeruginosa the potency of E-4695 was, in general, similar to that of ciprofloxacin and eightfold higher than those of ofloxacin and fleroxacin. E-4695 was four- and eightfold more potent than ciprofloxacin against C. perfringens and B. fragilis isolates, respectively. E-4695 and ciprofloxacin showed similar properties when the effects of pH or magnesium concentration were tested on them. E-4695 and ciprofloxacin had substantial reductions of activity only when pH decreased below 4.8. E-4695 and ciprofloxacin activities were not markedly affected by the presence of 5 or 10 mM Mg2+. The presence of serum and human urine at pH 7.2 decreased the activity of E-4695 between two- and fourfold. After an oral dose of 50 mg/kg of body weight, the maximum level in serum, the biological half-life, and the area under the concentration-time curve from 0 to 10 h for E-4695 were 13.2 microgram/ml, 3.3 h, and 45.6 microgram . h/ml, respectively. The area under the concentration-time curve from 0 to 4 h for ciprofloxacin was 2.3 microgram . h/ml at the same dose. Fifty-percent effective doses (ED50S) against Staphylococcus aureus HS-93 infections in mice were 4.5 mg/kg with E-4695 and 37.6 mg/kg with ciprofloxacin. Infection with Streptococcus pneumoniae 29206 was more effectively treated with E-4695 (ED50, 41,2 mg/kg) than with ciprofloxacin (ED50, 200 mg/kg). The ED50 of E-4695 for infections with Streptococcus pneumoniae 1625 was 132.2 mg/kg; ciprofloxacin was ineffective at 400 mg/kg against this strain. E-4695 was also more potent than ciprofloxacin in treatment of infections caused by gram-negative organisms such as Escherichia coli HM-42 (ED50S, 1.0 and 3.9 mg/kg, respectively). The ED50S of E-4695 and ciprofloxacin were 33.0 and 145.5 mg/kg against P. aeruginosa HS-116 and 9.6 and 18.9 mg/kg against P. aeruginosa B-120, respectively. The therapeutic efficacy of E-4695 may depend not only on its in vitro activity but also on its improved pharmacokinetic properties.

Animals↗
PubMed1994

Induction of heat shock proteins by abnormal proteins results from stabilization and not increased synthesis of sigma 32 in Escherichia coli.

Accumulation of abnormal proteins in cells of bacteria or eukaryotes can induce synthesis of a set of heat shock proteins. We examined such induction following addition of azetidine (a proline analog) or synthesis of a heterologous protein (human prourokinase) in Escherichia coli. Synthesis of heat shock proteins under these conditions increased almost immediately and continued with increasing rates until it reached a maximum after 30 to 60 min at 30 degrees C. The induction was closely accompanied by an increase in the cellular level of sigma 32 specifically required for transcription of heat shock genes. The increase in sigma 32 initially coincided with increased synthesis of heat shock proteins but then exceeded the latter, particularly following accumulation of prourokinase. The sigma 32 level increase upon either treatment was found to result solely from stabilization of sigma 32, which is ordinarily very unstable, and not from increased synthesis of sigma 32. This is in contrast to what had been found when cells were exposed to a higher temperature, at which both increased synthesis and stabilization of sigma 32 contributed to the increased sigma 32 level. On the basis of these and other findings, we propose that abnormal proteins stabilize sigma 32 by a pathway or a mechanism distinct from that used for the induction of sigma 32 synthesis known to occur at the level of translation. Evidence further suggests that the DnaK chaperone plays a crucial regulatory role in induction of the heat shock response by abnormal proteins.

Azetidines↗
PubMed1993

The DNA-binding activity of the human heat shock transcription factor is regulated in vivo by hsp70.

The human heat shock transcription factor (HSF) is maintained in an inactive non-DNA-binding form under nonstress conditions and acquires the ability to bind specifically to the heat shock promoter element in response to elevated temperatures or other conditions that disrupt protein structure. Here we show that constitutive overexpression of the major inducible heat shock protein, hsp70, in transfected human cells reduces the extent of HSF activation after a heat stress. HSF activation was inhibited more strongly in clones that express higher levels of hsp70. These results demonstrate that HSF activity is negatively regulated in vivo by hsp70 and suggest that the cell might sense elevated temperature as a decreased availability of hsp70. HSF activation in response to treatment with sodium arsenite or the proline analog azetidine was also depressed in hsp70-expressing cells relative to that in the nontransfected control cells. As well, the level of activated HSF decreased more rapidly in the hsp70-expressing clones when the cells were heat shocked and returned to 37 degrees C. These results suggest that hsp70 could play an active role in the conversion of HSF back to a conformation that does not bind the heat shock promoter element during the attenuation of the heat shock response.

Arsenic↗
PubMed1997

Induction of the mammalian GRP78/BiP gene by Ca2+ depletion and formation of aberrant proteins: activation of the conserved stress-inducible grp core promoter element by the human nuclear factor YY1.

Previously, we have identified a constitutive nuclear factor, p70CORE, from HeLa cell nuclear extract which interacts specifically with the stress-inducible change region (SICR) of the grp78 promoter. Here we report that p70CORE is identical to YY1, a member of the GLI zinc finger family, by criteria of biochemical properties including apparent molecular weight, binding site homology, immunoreactivity, and affinity purification. Recombinant YY1 binds the double-stranded SICR with high specificity but has no affinity for its single-stranded form. In cotransfection studies, YY1 specifically enhanced the transcriptional activation of the grp78 promoter under a variety of stress conditions: depletion of the endoplasmic reticulum calcium stores, protein glycosylation block, and formation of aberrant proteins by azetidine treatment. In contrast, YY1 has minimal effect on the stress induction of the hsp70 promoter. YY1 enhancement of the grp78 stress response is dependent on its DNA-binding domain, with little effect on the basal expression of the promoter. The effect of YY1 transactivation may be mediated by the highly conserved grp78 core element. This is the first example of the ubiquitous factor YY1 involved in regulating inducible gene expression and its involvement in mediating stress signals generated from the endoplasmic reticulum to the nucleus.

Azetidines↗
PubMed2001

[Development of aziridination and azetizination reactions of amino allenes using a palladium catalyst].

Palladium-catalyzed synthesis of alkenylaziridines and azetidines from alpha- and beta-amino allenes are presented. Whereas palladium-catalyzed reaction of N-arylsulfonyl-alpha-amino allenes with an aryl iodide in the presence of potassium carbonate in DMF at around 70 degrees C affords the corresponding 3-pyrroline derivatives, the reaction in refluxing 1,4-dioxane under similar conditions yields exclusively or most predominantly the corresponding 2-alkenylaziridines bearing an aryl group on the double bond. Similarly, N-arylsulfonyl-beta-amino allenes can be also cyclized into the corresponding alkenylazetidines bearing 2,4-cis-sunstituents under palladium-catalyzed cyclization conditions in DMF.

Alkadienes↗
PubMed2001

An orally active chymase inhibitor, BCEAB, suppresses heart chymase activity in the hamster.

We investigated the effects of a novel chymase inhibitor, BCEAB (4-[1-[[bis-(4-methyl-phenyl)-methyl]-carbamoyl]-3-(2-ethoxy-benzyl)-4-oxo-azetidine-2-yloxy]-benzoic acid). The IC50 value of BCEAB for purified human chymase was 5.4 nM, whereas BCEAB did not inhibit the angiotensin-converting enzyme, elastase and tryptase. In isolated dog arteries, the IC50 value of BCEAB for the angiotensin I-induced contraction in the presence of 1 microM lisinopril was 2.8 microM. In the hamster, the heart chymase activities were significantly suppressed to 42.0% and 26.9% 3 h after oral administration of 100 and 300 mg of BCEAB/kg of body weight, respectively. In conclusion, BCEAB is a useful chymase inhibitor for studying the role of chymase in vivo.

Angiotensin II↗
PubMed2002

Development of a chymase inhibitor: pharmacological characterization of a chymase inhibitor in inflamed tissue remodeling and fibrosis.

Chymase, a chymotrypsin-like serine protease, has not only alternative angiotensin II-generating activity but also various activities involving inflammatory responses. However, little is known of its contribution to physiological functions. Therefore, chymase inhibitors are thought to be potentially useful as tools for elucidating the physiological functions of chymase and therapeutic agents. Within the last five years, many patents on non-peptide chymase inhibitors have been published. We developed a potent non-peptide chymase inhibitor BCEAB (4-[1-[[bis-(4-methyl-phenyl)-methy]-carbamoyl]-3-(2-ethoxy-benzyl)-4-oxo-azetidine-2-yloxy]-benzoic acid) and examined its effect on inflamed tissue remodeling and fibrosis using a hamster sponge implant model. BCEAB has high inhibitory activity against human chymase but not against angiotensin-converting enzyme, elastase and tryptase. In the hamster sponge implant model, oral administration of BCEAB for 15 days dose-dependently suppressed both the dry weight of granuloma tissues in the sponge discs and the amounts of hydroxyproline in the tissues gradually increased during the experimental period. These results suggest that chymase, at least in part, participates in the growth of granuloma tissues of inflammatory regions by stimulating fibroblast growth and extracellular matrix collagen deposition. Chymase inhibitors for oral administration, such as BCEAB, might be useful for clarifying the pathophysiological roles of chymase in vivo.

Administration, Oral↗
PubMed2002

Differential accumulation of U14 snoRNA and hsc70 mRNA in Chinese hamster cells after exposure to various stress conditions.

We have previously characterized the unique organization of the U14 small nucleolar ribonucleic acid (snoRNA) gene in Chinese hamster HA-1 cells. The single copy of the hsc70/U14 gene is the only source for the production of both U14 snoRNA species and hsc70 messenger ribonucleic acid (mRNA) in these cells. Here we report that the accumulations of U14 snoRNA and hsc70 mRNA are different in response to various stress conditions, although both of them are transcribed in a single primary transcript. Heat shock induced an increased accumulation of both U14 snoRNA and hsc70 mRNA. On the other hand, exposure to sodium arsenite or azetidine induced an increased accumulation of hsc70 mRNA, but did not lead to a concomitant increase in the level of U14 snoRNA. Under normal growth conditions, the variations in the levels of U14 snoRNA and hsc70 mRNA, in the different phases of the cell cycle, are correlated. The increased expression of U14 snoRNA and hsc70 mRNA, and the hsc70 protein induced specifically by heat shock suggest that they participate in the repair process of heat-induced damage to macromolecular complexes involved in the synthesis and processing of ribosomal RNA.

3T3 Cells↗
PubMed2000

beta-Lactam derivatives as enzyme inhibitors: N-substituted derivatives of (S)-4-oxoazetidine-2-carboxylate as inhibitors of elastase and papain.

N-Alkyl and N-acyl substituted 4-oxoazetidine-2-carboxylates are synthesized and evaluated as inhibitors of the proteases porcine pancreatic elastase (PPE) and papain. The compounds are obtained by alkylation or acylation at the nitrogen of benzyl (S)-4-oxoazetidine-2-carboxylate, which is synthesized by a modified literature procedure. The enzymatic assays prove some derivatives to be effective inhibitors of PPE and/or papain. The N-BOC protected amino acid derivatives 10 and 13 inhibit PPE reversibly with KI-values in the micromolar range. On the other hand, papain is inactivated irreversibly by benzyl (S)-2-(benzyloxycarbonyl)azetidin-1-acetate (6).

Animals↗
PubMed1988

Site-specific alteration of cysteine 281, cysteine 344, and cysteine 349 in the proline carrier of Escherichia coli.

Cys-281, Cys-344, or Cys-349 in the proline carrier of Escherichia coli was changed to a serine residue by site-specific mutagenesis. The activities of the resultant mutants for uptake of proline were as great as that of the wild-type strain. These mutant carriers were all as sensitive as the wild-type carrier to the proline analogue azetidine 2-carboxylate. However, the mutant carriers with Ser-281 and Ser-344 were resistant to N-ethylmaleimide, whereas the mutant carrier with Ser-349 was as sensitive as the wild-type carrier to this reagent. These results indicate that these cysteine residues are not essential for proline transport and that Cys-281 and Cys-344 may be close to the substrate-binding site that contains an N-ethylmaleimide-sensitive residue.

Amino Acid Transport Systems, Neutral↗
PubMed1980

An experimental analysis of somite segmentation in the chick embryo.

In a previous paper it was suggested that collagen fibrils play an important role in the process of somite segmentation. This paper was designed mainly to test that concept. In one series of experiments, embryos were treated with either alpha, alpha'-dipyridyl or L-azetidine-2-carboxylic acid, which are analogues that interfere with the formation of normal collagen. The reagents led to a reduction in the numbers of somites that formed, as well as to the production of other anomalies such as overall diminution in size and retardation. The older the embryo at the time of treatment, the further posteriorly were the major anomalies located. It is concluded that these results lend some support to the concept. In a second series of experiments an incision was made along one side of the neural tube and notochord to separate it from the segmental plate on one side. The result was that many more somites formed on the unoperated (control) side of the embryo than on the operated side. It is concluded that these results also lend support to the concept; but that they are of interest also in relation to the mechanisms involved in the control of somite numbers. In a third group of experiments, attempts were made to obtain somites in the absence of endoderm. Although this was not possible using surgery, it was achieved by treating the young embryos with U.V. irradiation. It was concluded that the presence of endoderm is not essential for the segmentation of mesoderm.

2,2'-Dipyridyl↗
PubMed1994

Dipeptidyl peptidase IV: development, design, synthesis and biological evaluation of inhibitors.

Dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5) is a highly specific serine protease which cleaves off N-terminal dipeptides from peptides with a penultimate proline or alanine. The enzyme is widely distributed in mammalian cells and tissues, but specific activities differ greatly. In the hematopoietic system it is found almost exclusively on T cells, where it is identified as CD26, a T cell activation molecule. DPP IV may be involved in the metabolism of peptides, intestinal assimilation and cell adhesion and it plays an integral role in T cell activation. DPP IV inhibitors may provide help during the further elucidation of the biological function(s) of this enzyme. Moreover, because of the integral role the enzyme plays in T cell activation, specific inhibition of DPP IV may constitute a new way of immune modulation. N-Peptidyl-O-acylhydroxylamines and boronic acid analogues of proline and alanine are two known DPP IV inhibitors. The major drawbacks for their therapeutic use are for the hydroxylamines, the toxicity and for the boronic acid derivatives, the chemical liability. A low toxicity, acceptable stability and a high specificity are essential criteria for the design of inhibitors that are suitable, not only for experimental, but also for therapeutic use. Therefore we proposed 5 types of potential DPP IV inhibitors: azapeptides, azetidines, Michael substrates, reduced peptides and phosphonic acids. All the synthesized compounds possess a substrate-like structure, which is a pre-requisite for recognition by the enzyme. We choose for a modified proline or alanine at the penultimate position, substituted with glycine, alanine, valine, isoleucine or phenylalanine at the N-terminus. The prepared compounds were screened biologically at a 5 mM concentration with a fluorometric method using Gly-Pro-4-Me-2NA as substrate.

Aza Compounds↗
PubMed1998

Glutamatergic and GABAergic agonists increase [Ca2+]i in avian cochlear nucleus neurons.

Neurons of the avian cochlear nucleus, nucleus magnocellularis (NM), are stimulated by glutamate, released from the auditory nerve, and GABA, released from both interneurons surrounding NM and from cells located in the superior olivary nucleus. In this study, the Ca2+ indicator dye Fura-2 was used to measure Ca2+ responses in NM stimulated by glutamate- and GABA-receptor agonists using a chicken brainstem slice preparation. Glutamatergically stimulated Ca2+ responses were evoked by kainic acid (KA), alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA), and N-methyl-D-aspartate (NMDA). KA- and AMPA-stimulated changes in [Ca2+]i were also produced in NM neurons stimulated in the presence of nifedipine, an L-type Ca2+ channel blocker, suggesting that KA- and AMPA-stimulated changes in [Ca2+]i were carried by Ca2(+)-permeable receptor channels. Significantly smaller changes in [Ca2+]i were produced by NMDA. When neurons were stimulated in an alkaline (pH 7.8) superfusate, NMDA responses were potentiated. KA- and AMPA-stimulated responses were not affected by pH. Several agents known to stimulate metabotropic receptors in other systems were tested on NM neurons bathed in a Ca2+ free-EGTA--buffered media, including L-cysteine sulfinic acid (L-CSA), trans-azetidine dicarboxylic acid (t-ADA), trans-aminocyclo-pentanedicarboxylic acid (t-ACPD), and homobromoibotenic acid (HBI). The only agent to reliably and dose-dependently increase [Ca2+]i was HBI, an analog of ibotenate. GABA also stimulated increases in [Ca2+]i in NM neurons. GABA-stimulated responses were reduced by agents that block voltage-operated channels and by agents that inhibit Ca2+ release from intracellular stores. Whereas GABA-A receptor agonist produced increases in [Ca2+]i GABA-B and GABA-C receptor agonists had no effect. There appear to be several ways for [Ca2+]i to increase in NM neurons. Presumably, each route represents a means by which Ca2+ can alter cellular processes.

Animals↗
PubMed1985

Synthesis of collagen: chemical regulation of post-translational events.

Collagen biosynthesis involves many unique post-translational events. Inhibition of some of these will lead either to decreased formation of the extracellular collagen fibres or to an accumulation of fibres with altered functional properties. The events that would seem most suitable targets for chemical regulation are triple helix formation, the cleavage of propeptides from the procollagen molecules and cross-link formation. Attempts have recently been made to develop inhibitors of prolyl 4-hydroxylase in particular, as inhibition of this enzyme will prevent triple helix formation and thus lead to a non-functional protein. Prolyl 4-hydroxylase is inhibited competitively with respect to ferrous ion by several bivalent cations, especially zinc, with respect to 2-oxoglutarate by pyridine 2,5-dicarboxylate, pyridine 2,4-dicarboxylate, 3,4-dihydroxybenzoate and many related compounds, with respect to oxygen by superoxide dismutase-active copper chelates and with respect to the peptide substrate by a number of peptides. Triple helix formation can also be inhibited by administering certain proline analogues such as cis-4-hydroxyproline and L-azetidine-2-carboxylic acid, which are incorporated into proteins in place of proline. Only preliminary data are available on the possibilities for using any of these substances to inhibit collagen accumulation in fibrotic processes.

Animals↗
PubMed2003

Structure-activity relationships of dynorphin analogs substituted in positions 2 and 3.

Following up on the observation that the dynorphin analog [Pro(3)]Dyn A(1-11)-NH(2) 2 possesses high affinity and selectivity for the kappa opioid receptor, a number of related peptides were prepared and characterized by radioligand binding and [(35)S]GTPgammaS assays. While incorporation of 2-azetidine carboxylic acid in position 3 led to the equally potent analog 3, the corresponding analog containing piperidine-2-carboxylic acid showed a nearly 90-fold reduction in kappa affinity. Differential preferred bond angles phi in the three building blocks might account for these observations. Compounds 2 and 3 were kappa antagonists with IC(50) values of 380 and 350 nM, respectively. The Sar(3) analog 7 and the Sar(2) analog 8 were kappa agonists, with greater selectivity than Dyn A(1-11)-NH(2) 1. In view of their high kappa affinities (8: K(i) = 1.5 nM; 2: K(i) = 2.4 nM), the new analogs were surprisingly weak kappa agonists or antagonists, e.g., the EC(50) value for the agonist 8 was 280 nM. Different kappa receptor subtypes in binding vs functional assays can not account for these results, since both assays were performed using the same membrane preparation.

Amino Acid Sequence↗
PubMed2006

Old molecules for new receptors: Trp(Nps) dipeptide derivatives as vanilloid TRPV1 channel blockers.

The transient receptor potential vanilloid member 1 (TRPV1), an integrator of multiple pain-producing stimuli, is regarded nowadays as an important biological target for the discovery of novel analgesics. Here, we describe the first experimental evidence for the behavior of an old family of analgesic dipeptides, namely Xaa-Trp(Nps) and Trp(Nps)-Xaa (Xaa=Lys, Arg) derivatives, as potent TRPV1 channel blockers. We also report the synthesis and biological investigation of a series of new conformationally restricted Trp(Nps)-dipeptide derivatives with improved TRPV1/NMDA selectivity. Compound 15 b, which incorporates an N-terminal 2S-azetidine-derived Arg residue, was the most selective compound in this series. Collectively, a new family of TRPV1 channel blockers emerged from our results, although further modifications are required to fine-tune the potency/selectivity/toxicity balance.

Animals↗
PubMed1976

Evidence contrary to the protein error hypothesis for in vitro senescence.

A strain of diploid fibroblasts, obtained from the skin of a male infant, was cultured in vitro and cells were tested throughout their lifespan for the appearance of altered glucose-6-phosphate dehydrogenase (G-6-PD) detected either by thermostability studies or by immunotitration. No significant difference was found in the proportion of thermolabile enzyme in 31 young cultures (4.8 +/- 1%, S.E.), in comparison with that in 19 old cultures (4.9 +/- 1%, S.E.). Old cultures had ceased active cell division (49-60 doublings); DNA replication, measured by [3H]thymidine uptake over a period of 24 hours, was limited to less than 5% of these cells. Young cells (5-22 doublings) had a [3H]thymidine labeling index of 75-85%. Titration of G-6-PD activity in extracts of young and old cells with neutralizing antibody directes specifically against G-6-PD failed to detect an increment of enzymatically defective G-6-PD in old cells. The thermostability studies were capable of detecting altered G-6-PD in skin fibroblasts from a female heterozygous for a thermolabile mutant of G-6-PD, and in fibroblasts treated with a proline analogue, azetidine carboxylic acid. The immunotitration technique was also capable of detecting catalytically altered G-6-PD from the thermolabile mutant and G-6-PD inactivated with N-ethylameimide. These findings argue against a protein error catastrophe as the cause of in vitro clonal senescence.

Adult↗